Within nucleic acid and genetic material technologies, it is often necessary to determine whether a gene, a part of a gene, or a nucleotide sequence is present in a living organism, a cellular extract of this organism, or a biological sample. Since any gene or part of a gene is characterized by a specific sequence of nucleotide bases, it is only necessary to search directly for the presence of all or part of said specific sequence in a sample containing a mixture of polynucleotides.
There is enormous interest in this search for specific polynucleotide sequences, particularly in detection of pathogenic organisms, determination of the presence of alleles, detection of the presence of lesions in a host genome, or detection of the presence of a particular RNA or modification of a cell host. Genetic diseases such as Huntington's disease, Duchenne's disease, phenylketonuria, and beta thalassemia can thus be diagnosed by analyzing nucleic acids from the individual. Also it is possible to diagnose or identify viruses, viroids, bacteria, fungi, protozoans, or any other form of plant or animal life by tests employing nucleic probes.
Once the specific sequence of an organism or a disease is known, the nucleic acids should be extracted from a sample and a determination should be made as to whether this sequence is present. Various methods of nucleic acid detection have been described in the literature. These methods are based on the properties of purine-pyrimidine pairing of complementary nucleic acid strands in DNA-DNA, DNA-RNA, and RNA-RNA duplexes.
This pairing process is effected by establishing hydrogen bonds between the adenine-thymine (A-T) and guanine-cytosine (G-C) bases of double-stranded DNA; adenine-uracil (A-U) base pairs can also form by hydrogen bonding in DNA-RNA or RNA-RNA duplexes. The pairing of nucleic acid strands for determining the presence or absence of a given nucleic acid molecule is commonly called “nucleic acid hybridization” or simply “hybridization”.
The most direct method for detecting the presence of a target sequence in a nucleic acid sample is to obtain a “probe” whose sequence is sufficiently complementary to part of the target nucleic acid to hybridize therewith. A pre-synthesised probe can be applied in a sample containing nucleic acids. If the target sequence is present, the probe will form a hybridization product with the target. In the absence of a target sequence, no hybridization product will form. Probe hybridization may be detected by numerous methods known in the art. Commonly the probe may be conjugated to a detectable marker. Fluorescent or enzymatic markers form the basis of molecular beacons, Taqman and other cleavable probes in homogeneous systems. Alternatively the probe may be used to capture amplified material or labelled such that the amplicon is detected after separating a probe hybridized to the amplicon from non-hybridized material.
The main difficulty in this approach, however, is that it is not directly applicable to cases where the number of copies of the target sequence present in a sample is small, less than approximately 10′ copies. Under these conditions it is difficult to distinguish specific attachment of a probe to its target sequence from non-specific attachment of the probe to a sequence different from the target sequence. One of the solutions to this problem is to use an amplification technique which consists of augmenting the detection signal by a preliminary technique designed to specifically and considerably increase the number of copies of a target nucleic acid fragment if it is present in the sample.
The articles by Lewis (1992, Genetic Engineering News 12: 1-9) and Abramson and Myers (1993, Curr. Opin. Biotechnol. 4: 41-47) are good general surveys of amplification techniques. The techniques are based mainly on either those that require multiple cycles during the amplification process or those that are performed at a single temperature.
Cycling techniques are exemplified by methods requiring thermo-cycling and the most widely used of this class of technology is PCR (polymerase chain reaction, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; European Patent No. 0 201 184) which enables the amplification of a region of interest from a DNA or RNA. The method usually consists of three steps:
(i) dissociating (denaturing) a double-stranded DNA into single-stranded DNAs by heat denaturation/melting (FIG. 1B);
(ii) annealing a primer oligonucleotide to the single-stranded DNA (FIG. 1B); and
(iii) synthesizing (extending) a complementary strand from the primer in order to copy a region of a DNA of interest (FIG. 1C).
After this process is completed the system is heated which separates the complementary strands and the process is repeated. Typically 20-40 cycles are performed to amplify genomic DNA to the extent that it can be further analysed.
The majority of exponential nucleic acid amplification processes rely on an excess of upstream and downstream primers that bind to the extreme 3′ terminus and the complement of the extreme 5′ end of the target nucleic acid template under investigation as shown in FIGS. 1A-C.
A second class of amplification techniques, known as isothermal techniques, are those that are performed at a single temperature or where the major aspect of the amplification process is performed at a single temperature. In contrast to the PCR process where the product of the reaction is heated to separate the two strands such that a further primer can bind to the template repeating the process, the isothermal techniques rely on a strand displacing polymerase in order to separate/displace the two strands of the duplex and re-copy the template. This well-known property has been the subject of numerous scientific articles (see for example Y. Masamute and C. C. Richardson, 1971, J. Biol. Chem. 246, 2692-2701; R. L. Lechner et al., 1983, J. Biol Chem. 258, 11174-11184; or R. C. Lundquist and B. M. Olivera, 1982, Cell 31, 53-60). The key feature that differentiates the isothermal techniques is the method that is applied in order to initiate the reiterative process.
Broadly isothermal techniques can be subdivided into those methods that rely on the replacement of a primer to initiate the reiterative template copying (exemplified by HDA (Helicase Dependent Amplification), exonuclease dependent amplification (EP1866434), Recombinase Polymerase Amplification (RPA) and Loop Mediated Amplification (LAMP)) and those that rely on continued re-use or de novo synthesis of a single primer molecule (exemplified by SDA (Strand Displacement Amplification and nucleic acid based amplification (NASBA and TMA)).
Recombinase Polymerase Amplification (RPA) is a process in which recombinase-mediated targeting of oligonucleotides to DNA targets is coupled to DNA synthesis by a polymerase (Morrical S W et. Al. J Biol Chem. 1991 Jul. 25; 266(21):14031-8 and Armes and Stemple, U.S. application Ser. No. 10/371,641). WO 2008/035205 describes an RPA process of amplification of a double stranded target nucleic acid molecule, comprising the steps of: (a) contacting UvsX, UvsY, and gp32 proteins with a first and a second single stranded nucleic acid primer specific for said double stranded target nucleic acid molecule to form a first and a second nucleoprotein primer; (b) contacting the first nucleoprotein primer to said double stranded target nucleic acid molecule to create a first D loop structure at a first portion of said double stranded target nucleic acid molecule and contacting the second nucleoprotein primer to said double stranded target nucleic acid molecule to create a second D loop structure at a second portion of said double stranded target nucleic acid molecule such that the 3′ ends of said first nucleic acid primer and said second nucleic acid primer are oriented toward each other on the same double stranded target nucleic acid molecule without completely denaturing the target nucleic acid molecule; (c) extending the 3′ end of said first and second nucleoprotein primers with one or more polymerases capable of strand displacement synthesis and dNTPs to generate a first and second double stranded target nucleic acid molecule and a first and second displaced strand of nucleic acid; and (d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached.
In order to discriminate amplification of the target from that of futile amplification producing artefacts, probe based systems may be used that detect sequences of the amplicon under investigation that are not present in the primers supplied to the system.
All of these processes rely only on a template that comprises a binding site for the two primers at their extreme termini. A template with these qualities can be produced by non-specific interactions between the upstream and downstream primers alone and the product (primer-dimers) may be capable of efficient amplification independently of the template under investigation, as shown in FIGS. 1D-E. As a consequence of this futile amplification, the assay components become consumed by non-productive events limiting the sensitivity of the assay process.
It is an object of the present invention to provide an alternative isothermal nucleic acid amplification technique. It is a further object of the present invention to provide an exponential amplification technique. It is a further object to minimize or eliminate amplification artefacts and so provide a method for amplifying nucleic acids with increased specificity and/or sensitivity.